Recirculation container

ABSTRACT

A bag or reservoir for recirculation washing of blood cells having a top outlet port and bottom inlet port. A method of recirculation washing of blood cells using the bag in conjunction with a spinning membrane filter. The method can be used in an instrument for magnetic cell selection or a stand-alone cell washing apparatus.

BACKGROUND OF THE INVENTION

[0001] This invention relates to recirculation washing of blood cellsusing a spinning membrane filter, and in particular to recirculationwashing of blood cells in a magnetic cell selection apparatus.

[0002] Fischel U.S. Pat. No. 5,034,135, issued Jul. 23, 1991, andSchoendorfer U.S. Pat. No. 5,035,121, issued Oct. 1, 1991 disclosespinning membrane filters comprising a cylindrical housing andconcentric grooved cylindrical rotor. The rotor is covered with amembrane the membrane is spaced from the inner wall of the housing.Blood is introduced into the gap between the membrane and housing.Filtrate passes through the membrane, into the grooves of the rotor,into tubes which communicate with the grooves, and out the bottom centerof the spinning membrane filter. Concentrated cells are removed from thegap. FIGS. 7 and 8 in the Fischel patent illustrate a cell washingmodification in which a porous wall is interposed between the membraneand the inner wall of the housing. Blood is introduced into the gapbetween the membrane and the porous wall and an isotonic wash solutionis introduced into the gap between the porous wall and the inner wall ofthe housing. FIG. 6 in the Schoendorfer patent illustratse introductionof a rinse solution with the blood. Schoendorfer et al. U.S. Pat. No.5,035,121, issued Oct. 1, 1991, discloses use of two spinning membranefilters in series or parallel. A washing solution is introduced into atleast one of the spinning membrane filters.

[0003] Duff U.S. Pat. No. 5,234,608, issued Aug. 10, 1993, discloses aspinning membrane filter of the type which is preferred for use inconjunction with this invention. According to the disclosure, cell-richconcentrate is removed from the upper portion of the gap between themembrane and the inner wall of the housing, cell-poor plasma filtrate isremoved from the bottom center of the spinning membrane filter. Sourcecell suspension is mixed with cell-rich concentrate and introduced tothe lower portion of the gap area.

[0004] Schoendorfer et al. U.S. Pat. Nos. 4,675,106, issued Jun. 23,1987, U.S. Pat. No. 4,753,729, issued Jun. 28, 1988, and U.S. Pat. No.4,816,151, issued Mar. 28, 1989, disclose drive mechanisms for spinningmembrane filters.

[0005] Moubayed et al. U.S. Pat. No. 5,536,475 discloses asemi-automated instrument for selection of blood cells usingparamagnetic beads which are coated with a binding agent such as anantibody which binds specifically to the cells to be selected. Theinstrument comprises a primary magnet associated with a primarycontainer and a secondary magnet associated with a secondary container.Blood cells, liquid and beads are agitated in the primary container toform a conjugate between the beads and the selected cells. The primarymagnet is then moved into a position adjacent the primary container tomagnetically capture the bead/cell conjugate and the non-selected cellsand liquid are removed. The primary magnet is then moved into a positionaway from the primary container to release the bead/cell conjugate. Washsolution is added and the contents of the primary container areagitated, then the primary magnet is moved into the position adjacentthe primary container to again capture the bead/cell conjugate and thewash solution is removed. The primary magnet is again moved into aposition away from the primary container to release the bead/cellconjugate. Liquid containing a reagent which releases the selected cellsfrom the beads is added and the contents are again agitated. The primarymagnet is again moved into the position adjacent the primary containerto capture the beads. The released cells and liquid are introduced tothe secondary container. The secondary container is positioned adjacentto the secondary magnet to capture any beads which may have escaped theprimary magnet. The instrument is used with a disposable set comprisingplastic bags for wash liquid, cell suspension and bead suspension,interconnected with plastic tubing.

[0006] The semi-automated instrument disclosed in the Moubayed et al.patent is sold by Baxter Healthcare Corporation. under the trademarkIsolex® 300 SA. A modified version of the instrument is sold by theBaxter Healthcare Corporation under the trademark Isolex® 300i. The 300idiffers from the 300 SA in that it is fully automated and it includes aspinning membrane filter for washing the selected cells and also forremoving platelets from the source cells prior to selection.

[0007] Chapman et al. International Publication WO 95/13837, publishedMay 26, 1995, discloses a peristaltic pumping assembly of a type whichis used to move fluids in the Isolex® 300 SA and Isolex® 300iinstruments. Deniega et al. International Publication WO 95/14142,published May 26, 1995, discloses an organizer frame of a type which isused with the peristaltic pumping assembly in the Isolex® 300 SA andIsolex® 300i instruments. The organizer frame is also used on a machinefor separation of platelets from whole blood. Deniaga discloses a tubingset which includes a spinning membrane filter and a reservoir forplatelet-poor packed blood cells. The reservoir has a top and bottomport. Packed cells from the outlet of the spinning membrane filter enterthrough the top inlet port of the reservoir. Whole blood from a patiententers through the bottom inlet port.

[0008] Recirculation washing of selected blood cells is performed in theIsolex® 300i utilizing the spinning membrane filter in conjunction witha recirculation wash bag which has both inlet and outlet ports at thebottom and no port at the top. The bag is a 600 ml bag with the inletand outlet ports separated by about 2 inches. The bag has been able toconcentrate cell suspensions that normally start at about 400 ml. Thisbag performed better when it was occasionally massaged. This is the onlyway to process more than about 5×10¹⁰ cells in the bag.

[0009] The above-cited U.S. patents and International Publications areeach incorporated herein by reference.

SUMMARY OF THE INVENTION

[0010] This invention includes a method, a bag and a disposable set forrecirculation washing of blood cells. The invention can be used forwashing of blood cells in a magnetic cell selection instrument, but canalso be used for washing whole blood or other blood cell products.

[0011] The recirculation wash bag is a flexible plastic bag which has atop port and a bottom port. In one embodiment, an integral coarse filtercomprising a tube of semi-rigid plastic mesh extends from the top portinto the bag. This filter provides mild resistance to larger cellaggregates. In another embodiment, the bag includes a bubble trap at thetop comprising tubing extending into the bag from the top port. In thepreferred embodiment, the bag includes both the semi-rigid integralfilter and the bubble trap; the tubing for the bubble trap fits insidethe plastic mesh tube to provide a space to accumulate air around thetubing. When a system incorporating the bag is primed with buffersolution, vacuum is pulled on the bag. Because the filter is semirigid,it holds open a path through the otherwise collapsed bag for the cellsto move up to the top port.

[0012] The method of the invention utilizes a flexible plasticrecirculation wash bag and a spinning membrane filter. The spinningmembrane filter has an inlet port for a diluted suspension of bloodcells in buffer solution, a first outlet port for filtrate, and a secondoutlet port for a concentrated suspension of blood cells in buffersolution. The recirculation wash bag has a top outlet port and a bottominlet port. Preferably, the recirculation wash bag includes the integralcoarse filter and bubble trap described above.

[0013] The method comprises withdrawing a suspension of blood cells inbuffer solution from the recirculation wash bag through the top port,mixing the suspension with additional buffer solution to form a dilutedsuspension of blood cells in buffer solution, feeding the dilutedsuspension into the spinning membrane filter through the inlet port,withdrawing filtrate comprising buffer solution from the spinningmembrane filter through the first outlet port, withdrawing aconcentrated suspension of blood cells in buffer solution from thespinning membrane filter through the second outlet port, feeding theconcentrated suspension into the bag through the bottom port, andcontinuing the recirculation washing until the desired amount of washinghas been achieved. A method for determining when the desired amount ofwashing has been achieved, based on an estimate of “residual,” isdescribed below. The residual represents the target component forreduction (e.g., platelets, antibody, etc., as described below).

[0014] In one embodiment of the method, the suspension of blood cellswithdrawn through the top port of the recirculation wash bag is mixedwith unwashed blood cells as well as buffer solution before feeding thediluted suspension into the spinning membrane filter. In one aspect ofthis embodiment, the unwashed blood cells include platelets, thefiltrate comprises a suspension of platelets in buffer solution, and therecirculation washing is continued until the platelet content of theconcentrated suspension of cells has been reduced to the desired level.

[0015] In another embodiment of the method, the recirculation wash bagat the beginning of the recirculation wash procedure contains, inaddition to blood cells, an antibody which specifically binds an antigenon certain of the blood cells, the filtrate comprises a suspension ofthe antibody in the buffer solution, and the recirculation washingcontinues until the concentrated suspension of cells contains thedesired amount of excess, unbound antibody.

[0016] In another embodiment of the method, the recirculation wash bagat the beginning of the recirculation wash procedure contains bloodcells which have been selected in a magnetic cell selection procedureand a peptide release agent which was used to release the selected cellsfrom a cell/magnetic bead conjugate, the filtrate comprises a solutionof the peptide release agent in buffer solution, and the recirculationwashing is continued until the peptide release content of theconcentrated suspension of cells has been reduced to the desired level.

[0017] The disposable set of the invention comprises the recirculationwash bag and the spinning membrane filter having ports as describedabove, and a filtrate bag, plus associated tubing, including tubing fora buffer solution bag. Plastic tubing connects the top port of therecirculation wash bag to a mixing zone. Plastic tubing with a bufferbag spike coupler at one end is connected to the same mixing zone. Themixing zone is connected by plastic tubing to the inlet port of thespinning membrane filter. The first outlet port of the spinning membranefilter is connected by plastic tubing to the inlet port of the filtratebag. The second outlet port of the spinning membrane filter is connectedby plastic tubing to the bottom port of the recirculation wash bag.

[0018] The disposable set may also include other bags and associatedtubing for use in a magnetic cell selection instrument, such as a bagfor antibody suspension in buffer solution, a bag for peptide releaseagent solution in buffer solution, a bag for a suspension of thenon-selected cells in buffer solution, and an end product bag for washedcells. A bag for unwashed cells (also referred to as a cell source bag)and/or a bag for buffer solution may be included in the set, but in thepreferred embodiment these items are supplied separately.

[0019] Use of a flexible recirculation wash bag with ports at the topand bottom and flow from bottom to top provides several advantages ascompared to a bag with inlet and outlet ports at the bottom, ascurrently used on the Isolex® 300i. First, using a flexible bag allowsthe volume to be varied depending on the number of cells. Exiting fromthe top has the advantage of removing the less dense supernatantpreferentially. This aids in making the concentration ratio high. (Theimportance of high concentration ratio is discussed below). For largevolumes or slow flow rates, some sedimentation of the larger cells alsoaids in reducing the cell concentration at the outlet port. The systemhas the advantage of having the most washed and most concentrated cellsat the bottom with the least washed and least concentrated cells at thetop. Additional advantages include the following: (1) allows accurateresidual estimates which in turn allow optimal residual levels insteadof just reduction; (2) provides more uniform processing of cells whichleads to a more uniform product for the selection process; (3) manualmassaging of the bag during the wash is not required, permittinghands-free operation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 illustrates the preferred embodiment of the recirculationwash bag of this invention. In the description which follows therecirculation wash bag having the configuration shown in FIG. 1 isreferred to as the IsoFlow™ bag.

[0021]FIG. 2 illustrates a disposable set of this invention which isadapted for use on a magnetic cell selection device such as the Isolex®300i.

[0022]FIG. 3 illustrates a disposable cell wash set of the inventionwhich is adapted for use on a stand-alone cell washing apparatus.

DETAILED DESCRIPTION OF THE INVENTION

[0023] IsoFlow™ Recirculation Wash Bag

[0024] Referring to FIG. 1, the IsoFlow™ bag is indicated generally bythe numeral 5. The bag is made of a flexible plastic such as andincludes bottom port 1 and top port 2. An integral coarse filtercomprising a tube of semi-rigid plastic mesh 3 extends from the top portinto the bag to within about ½ to 3 inches, preferably about 1 inch,from the bottom of the bag. The mesh tube is about ½ to about 1.5 inchesin diameter, preferably about 1 inch in diameter, and is preferablyclosed at its lower end. The tube's mesh (opening) size is in the rangeof about 80-400 microns, preferably about 230 microns. The bag includesa bubble trap at the top which is created by inserting tubing 4 into thetop port about ½ to 3 inches, preferably about 1.5 inches. Suitablematerials of construction include polyvinyl chloride (PVC) for the bag,polyester (e.g. Cleartuf®, shell) for the mesh tube filter, and PVC forthe tubing. Volume of the bag can vary, but will generally be between100 and 1500 ml. As presently designed for use on the Isolex® 300i, thebag holds a volume of 400 ml. The mesh could be replaced by some othersemi-rigid, rigid or combination structure that facilitates flow frombottom to top.

[0025] Isolex® 300i Cell Washing System

[0026] Referring to FIG. 2, the disposable set of this inventioncomprises the IsoFlow™ bag 5 and spinning membrane filter 6 andassociated tubing, including tubing for connecting a bag containingbuffer solution. Spinning membrane filter 6 (sometimes referred tosimply as “spinning membrane” or “spinner”) has the construction shownin FIG. 2 of Duff U.S. Pat. No. 5,234,608. The membrane is a nominal 4micron polycarbonate membrane. The buffer solution bag is not shown, butis indicated at 7; it is a standard flexible plastic bag with a bottomoutlet port, and is supplied separately. The top port 2 of IsoFlow™ bag5 is connected by tubing 8 having a sampling device 8 a to the bottomright channel 9 b (indicated by dotted lines) of clamp manifold 9.Channel 9 b is a mixing zone for mixing cells from IsoFlow™ bag 5 withbuffer solution from bag 7 and (in the platelet separation stepdescribed below) with unwashed cells from bag 44. Channel 9 b of clampmanifold 9 is connected by tubing 10 to the inlet port 11 of spinningmembrane filter 6. The bottom port 1 of IsoFlow™ bag 5 is connected bytubing 12 to the bottom left channel of clamp manifold 9 and tubing 13connects the bottom left channel of clamp manifold 9 to the outlet port14 of spinning membrane filter 6. Tubing 15 connects the outlet port ofbuffer solution bag 7 to the top right channel of clamp manifold 16;tubing 17 connects the top right channel of clamp manifold 16 to thebottom left channel of clamp manifold 18; tubing 19 connects the bottomleft channel of clamp manifold 18 to the bottom right channel of clampmanifold 18 and tubing 20 connects the bottom right channel of clampmanifold 18 to the bottom right channel 9 b of clamp manifold 9. Tubing15 is connected to a buffer bag spike coupler 21 and a sterilizingfilter 22. Tubing 23 connects filtrate outlet port 24 of spinningmembrane filter 6 with the top right channel of clamp manifold 25.Tubing 26 connects the top right channel of clamp manifold 25 withY-connector 27. Tubing 28 connects Y-connector 27 to the inlet port 29of filtrate (waste) bag 30. On tubing 28 is a clamp 31. Tubing 32connects Y-connector 27 to Y-connector 33. Tubing 32 carries a clamp 40.Tubing 34 connects Y-connector 33 to inlet port 35 of waste bag 36.Tubing 37 connectes Y-connector 33 to inlet port 38 of waste bag 39.Tubing 41 connects the top right channel of clamp manifold 25 topressure transducer protector 42.

[0027] There are three configurations of clamp manifolds shown in FIG.2. All configurations have clamps capable of obstructing the tubing thatruns through them on a flat platen (not shown) in the center of themanifolds. The dotted lines in the upper and/or lower portions of theclamp manifolds indicate the locations of channels within the manifolds.The dotted lines in clamp manifold 45 show that the bottom channelconnects all 4 tubes. The dotted lines in clamp manifolds 9 and 18 showthat there are two bottom channels—the left channel connects the twoleft tubes and the right bottom connects the two right tubes. The dottedlines in clamp manifolds 16 and 25 show that the bottom left channelconnects the tubes on the left and the top right channel connects thetubes on the right.

[0028] In the preferred embodiment illustrated in FIG. 2, the disposableset of the invention also includes other bags and containers andassociated tubing adapted for use on a magnetic cell separationinstrument such as the the Isolex® 300i. Tubing 43 connects a cellsource bag (not shown, but indicated at 44) with the bottom channel ofclamp manifold 45. Tubing 46 connects the bottom channel of clampmanifold 45 with the bottom left channel 18 a of clamp manifold 18.Channel 18 a is a mixing zone for buffer from bag 7 and unwashed cellsfrom bag 44. Tubing 43 is connected to a starting cells spike coupler47.

[0029] Bag 48 is a bag for antibody which reacts specifically with cellsto be selected on the Isolex® 300i. For example, where CD34+ cells areto be selected, bag 48 will contain anti-CD34 antibody. The bag has aninjection site 49 for injection of the antibody solution and an outletport 50 connected to a sterilizing filter 51. Tubing 52 connectssterilizing filter 51 to the bottom channel of clamp manifold 45.

[0030] Bag 53 is a bag for a peptide release agent which displaces theantibody from the cells after the cells have been magnetically selected.Bag 53 has an injection site 54 a for injection of a solution of thepeptide and an outlet port 54 connected to a sterilizing filter 55.Tubing 56 connects sterilizing filter 55 to the bottom channel of clampmanifold 45.

[0031] Cylinder 57 is the primary magnet separation chamber. It has avent filter 59 and an injection site 58 for injection of paramagneticmicrobeads coated with an antibody which binds specifically to theantibody in bag 48. It has a bottom port 60 which serves as both inletand outlet for cell suspensions. In use it is mounted on a rockermechanism as described in Moubayed et al. U.S. Pat. No. 5,536,475. Port60 is connected by tubing 61 to the bottom left channel of clampmanifold 16. That channel is connected by tubing 62 to the right topchannel of clamp manifold 16. The top right channel of manifold 16 isconnected by tubing 72 to the top right chamber of clamp manifold 25.The bottom left channel of clamp manifold 16 is also connected by tubing63 to Y-connector 64 and the latter is connected by tubing 65 to thebottom channel of clamp manifold 45. Y-connector 64 is also connected bytubing 66 to a pressure transducer protector 67.

[0032] Bag 68 is the secondary magnet separation bag described inMoubayed et al. U.S. Pat. No. 5,536,475. It has inlet port 69 and outletport 70. Inlet port 69 is connected by tubing 71 to the bottom leftchannel of clamp manifold 18. Outlet port 70 is connected by tubing 73to the bottom right channel of clamp manifold 18.

[0033] Bag 74 is a selected cell wash bag. It has two bottom ports.Inlet port 75 is connected by tubing 77 which has a sampling device 77 ato the bottom right channel 9 b of clamp manifold 9. Outlet port 76 isconnected by tubing 78 to the bottom left channel of clamp manifold 9.If desired, an IsoFlow™ bag can be substituted for the selected cellwash bag.

[0034] Bag 79 is an end product bag. It has an injection site 80 and aninlet port 81. Tubing 82 carrying sampling device 82 a and clamp 83connects inlet port 81 with the bottom channel of clamp manifold 45.

[0035] Frame 84 is an organizer frame as described in Denieaga et al.International Publication WO 95/14142 for use with a peristaltic pumpassembly (not shown) as described in Chapman et al. InternationalPublication WO 95/13837. Tubing 13, 15, 26 and 46 each passes throughone of the four pumping modules of the peristaltic pump assembly.

[0036] The volume of bags can vary, depending upon the volume of cellsto be processed. In the the commercial Isolex® 300i instrument, each ofbags 30, 36 and 39 has a volume of 2000 ml, each of bags 48, 53 and 79has a volume of 150 ml, and bag 74 has a volume of 600 ml. For use inthis system, the IsoFlow™ bag 5 has a volume of 400 ml.

[0037] At the beginning of a cell selection procedure, the disposableset of FIG. 2 is placed on the Isolex 300i. Bag 7 containing buffer andbag 44 containing source cells are attached. The source cells aretypically a leukapheresis product from a cell separation device such asa Fenwall 3000 CS. The buffer bag has a capacity of 4000 ml and astarting volume of at least 3500 ml. The cell source bag has a capacityof 1000 ml and a starting volume of about 500 ml. By appropriateoperation of clamps in the clamp manifolds and the pumps on tubing 13,15, and 46, buffer solution is added to the following elemments andconnecting tubing to prime the system: Isoflow™ bag 5, secondary magnetpouch 68, spinning membrane filter 6, filtrate bag 30, selected cellwash bag 74, release agent bag 53, antibody bag 48, cell source bag 44.During the prime, fluid is added to the Isoflow™ bag, the air is removedfrom the top part of the bag, more fluid is added through the bottompart, and excess air is released through tubing 8 to waste bag 30.

[0038] At this point the system is ready for removal of platelets fromthe leukapheresis product in cell source bag 44, using the method ofthis invention. For purpose of the following description: clamps inclamp manifold 45 are designated clamps C1, C2, C3, C4; clamps in clampmanifold 9 are designated C5, C6,C7, C8; clamps in clamp manifold 16 aredesignated C9, C10, C11, C12; clamps in clamp manifold 18 are designatedC13, C14, C15, C16; clamps in clamp manifold 25 are designated C17, C18,C19, C20; the pump on tubing 46 is designated P1, the cell source pump;the pump on tubing 15 is designated P2, the buffer pump; the pump ontubing 13 is designated P3, the recirculation pump; the pump on line 26is designated P4, the filtrate pump; and the rotor of spinning membranefilter 6 is designated as pump PS.

[0039] Prior to beginning cell wash, clamps C6, C8, C10, C11, C12, C14,C16 and C20 are opened, pumps P2, P3, P4 and P5 are moving. Thiscirculates buffer solution from bag 7, into the inlet port 11 and out ofoutlet ports 14 and 24 of spinning membrane filter 6, into bottom port 1and out of top port 2 of IsoFlow™ bag 5, and into filtrate bag 30.

[0040] To conduct recirculation washing of the blood cells for plateletremoval, clamps C1, C6, C8, C12, C14, C16 and C20 are open, pumps P1,P2, P3, P4, and PS are moving. A suspension of unwashed blood cells iswithdrawn from cell source bag 44 through tubing 43 to the bottomchannel of clamp manifold 45, then out through tubing 46 to the bottomleft channel 18 a of clamp manifold 18 where it is mixed with buffersolution. The buffer solution is withdrawn from buffer bag 7 throughtubing 15 to the top right channel of clamp manifold 16, then outthrough tubing 17 to the bottom left channel 18 a of clamp manifold 18.The diluted suspension of blood cells in buffer solution flows out ofthe bottom left channel 18 a through tubing 19 into the bottom rightchannel of clamp manifold 18, then out through tubing 20 to the bottomright channel 9 b of clamp manifold 9, where it is mixed with additionalbuffer solution from top port 2 of Isoflow™ bag 5. The dilutedsuspension of blood cells in buffer solution flows from channel 9 bthrough tubing 10 to the inle port 11 of spinning membrane filter 6.Platelets, a few red cells, and buffer flow through the membrane and outthrough outlet port 24 through tubing 23 to the top right channel ofclamp manifold 25, then out through tubing 26 and 28 to filtrate bag 30(clamp 31 open, clamp 40 closed). (The nominal 4 miron membrane usedremoves about 95% of platelets from a leukapheresis product, while about50% of red cells are also removed.) A concentrated suspension of bloodcells in buffer flows from the exit port 14 of spinning membrane filter6 through tubing 13 to the bottom left channel of clamp manifold 9, thenout through tubing 12 through the bottom port 1 into Isoflow™ bag 5. Asthe process continues, a suspension of blood cells in buffer solutionflows out of the top of the Isoflow™ bag 5. These cells are mixed inmixing zone 9 b with unwashed cells from source bag 44 and arerecirculated through the spinning membrane filter 6. Recirculationwashing is continued until the desired level of platelet removal hasbeen achieved.

[0041] After platelet removal, antibody in buffer solution istransferred to the concentrated suspension of blood cells in buffersolution in the Isoflow™ bag 5. For transfer of antibody solution frombag 48 to Isoflow™ bag 5, clamps C3, C6, C8, C14, C16 and C20 are openand pumps P1, P3 and P5 are moving. The antibody and cells are mixed inmixing zone 9 b. Then the antibody tubing is rinsed with buffer solutionwhile the antibody/cell suspension circulates through the Isoflow™ bag 5and spinning membrane filter 6. This occurs with clamps C6, C8, C10,C11, C14, C16 and C20 open,and with pumps P1, P2, P3 and P5 moving. Nextthe antibody/cell suspension is circulated through the IsoflowTm bag 5and spinning membrane filter 6 to sensitize the cells by binding withthe antibody. This is accomplished with clamps C6, C8 and C20 open, andwith pumps P3 and P5 moving.

[0042] After the cells have been sensitized by binding with antibody,they are washed to remove excess unbound antibody using the method ofthis invention. With clamps C6, C8, C12, C14, C16 and C20 open and withpumps P2, P3, P4 and P5 moving, a suspension of blood cells in buffersolution and containing excess unbound antibody is withdrawn fromIsoflow™ bag 5 through top port 2 and flows through tubing 8 to themixing zone 9 b in clamp manifold 9. Buffer solution is withdrawn frombuffer bag 7 through tubing 15, clamp manifold 16, tubing 17, clampmanifold 18 (left channel), tubing 19, clamp manifold 18 (right channel)and tubing 20, as previously described, to mixing zone 9 b, where it ismixed with the suspension of blood cells from Isoflow™ bag 5 to form adiluted suspension of blood cells containing excess unbound antibody.This diluted suspension flows through tubing 10 to inlet port 11 of thespinning membrane filter 6. Filtrate comprising antibody in buffersolution flows out of outlet port 24, through tubing 23, clamp manifold25, tubing 26, tubing 28, and port 29 into filtrate bag 30. Aconcentrated suspension of blood cells in buffer solution flows from theoutlet port 14 of the spinning membrane filter 6, through tubing 13,clamp manifold 9 (bottom left channel), tubing 12 and bottom port 1 intoIsoflow™ bag 5. The recirculation washing is continued until the cellsuspension contains the desired level of unbound antibody.

[0043] After antibody sensitization and removal of excess unboundantibody, the cells are transferred to primary magnet separation chamber57. Antibody-coated paramagnetic microbeads are mixed with the cells toform a conjugate between the microbeads and the sensitized cells, theconjugate is magnetically separated from the non-sensitized cells, thenon-sensitized cells are transferred to waste bag 36, peptide releaseagent from bag 53 is added to the chamber 57 to release the selectedcells, the selected cells are transferred to the secondary magnetseparation bag where any remining microbeads are separated magnetically,and the selected cells are transferred to selected cell wash bag 74. Theselected cells are then recirculation washed to remove excess peptiderelease agent using spinning membrane filter 6, all in conventionalmanner. If desired, selected cell wash bag can be an Isoflow™ bag, andthe recirculation wash to remove peptide release agent can be conductedusing the method of this invention. After removal of peptide releaseagent, the selected cells are transferred to end product bag 79.

[0044] Stand-Alone Cell Washing System

[0045]FIG. 3 illustrates a disposable set of the invention which isadapted for use on a standalone cell washing apparatus, i.e., anapparatus which does not include a cell selection function such as themagnetic cell selection of the Isolex® 300i instrument.

[0046] The disposable set includes Isoflow™ bag 5 having top port 2 andbottom port 1, spinning membrane filter 6 having inlet port 11 for adiluted suspension of blood cells, outlet port 14 for a concentratedsuspension of blood cells, and outlet port 24 for filtrate, and filtratebag 30 having inlet port 29. It may also include one or more of washedcell bag 79 having outlet port 81, unwashed cell bag 44 having outletport 47, and buffer solution bag 7 having outlet port 21. Top port 2 ofIsoflow™ bag 5 is connected by tubing 8 to connector 89. Port 21 ofbuffer bag 7 is connected by tubing 15 to Y-connector 95 and the latteris connected by tubing 20 carrying clamp C1 to connector 89. Port 47 ofunwashed cell bag is connected by tubing 43 carrying clamp C3 toY-connector 93 and then by tubing 91 to connector 89. Connector 89serves as a mixing zone for unwashed cells in buffer solution from bag44, recirculating cells in buffer solution from bag 5 and buffersolution from bag 7. Connector 89 is connected by tubing 10 to inletport 11 of spinning membrane filter 6. Filtrate outlet port 24 ofspinner 6 is connected by tubing 23 to Y-connector 94 and by tubing 26to the inlet port 29 of filtrate bag 30. Connector 95 is connected bytubing 92 carrying clamp C2 to connector 94. Connector 94 is connectedby tubing 41 to pressure transducer 90. Oulet port 14 of spinner 6 isconnected by tubing 13 to the bottom port 1 of Isoflow™ bag 5.Y-connector 93 is connected by tubing 82 carrying clamp C4 to inlet port81 of washed cell bag 79.

[0047] During recirculation washing, a suspension of blood cells inbuffer solution is withdrawn from the Isoflow™ bag 5 through the topport 2 and flows through tubing 8 to mixing zone 89. Unwashed cells inbuffer solution are withdrawn from bag 44 through port 47 and (withclamp C3 open and clamp C4 closed) through tubing 43 to Y-connector 93and then through tubing 91 to mixing zone 89 by the transfer pump P2.Buffer solution is withdrawn from bag 7 through port 21 and tubing 15 toconnector 95 by the buffer pump P2. With clamp C1 open, buffer flowsthrough tubing 20 to mixing zone 89. A diluted suspension of blood cellsin buffer solution flows from mixing zone 89 through tubing 10 to inletport 11 of spinner 6. A concentrated suspension of blood cells in buffersolution flows through outlet port 14 of spinner 6 through tubing 13 andinlet port 1 into Isoflow™ bag 5 by recirculation pump P3. Filtrateflows through outlet port 24 in spinner 6 and tubing 23 to connector 94and, with clamp C2 closed, through tubing 26 and inlet port 29 intofiltrate bag 30 by pump P4. Recirculation washing is continued until thedesired amount of target component has been removed from the bloodcells. Clamps C1, C2 and C3 are then closed, clamp C4 is opened, and thedirection of pump P1 is reversed, so that the suspension of washed cellsflows from bag 5 through tubing 8, 91 and 82 and port 81 into washedcell bag 79. The lines, bag and spinner are then rinsed by closingclamps C1 and C3, opening clamps C4 and C2, and pumping buffer with pumpP2 in series with pumps P1 and P3 to rinse the spinner, Isoflow™ bag andtubing.

[0048] System Controls

[0049] In carrying out the recirculation washing method of thisinvention, the filtrate rate (f) is typically fixed at about 70 ml/min.During the transfer of cells into the wash circuit, the recirculationrate (r) provides the primary pressure regulation (using theconcentration ratio CR described below) and varies from 14 to 70 ml/min.During the recirculation phase the recirculation rate ranges from about24 to 70 ml/min. The buffer solution rate (b) ranges from 0 to 70ml/min. to maintain a minimum scale volume and as a secondary pressureregulation mechanism. The rotor of the spinning membrane filter operatesat a maximum of 3700 RPM and a minimum of about 2340 RPM during normalprocessing.

[0050] The Isolex® 300i system is automatically controlled usingmicroprocessors. These microprocessors in-turn control 5 banks of 4clamps each (clamps C1-C20), 1 bank of pumps (pumps P1 -P4), 1 spinnermotor drive P5 (drive for the rotor of spinning membrane filter 6), and1 rocker assembly for container 57 with an integral magnet carriage tofacilitate separation of magnetic beads (not shown, but described inMoubayed et al. U.S. Pat. No. 5,536,475). The system uses feedback from6 weight scales (not shown), 2 pressure transducers (not shown, butattached to line 66 at 67 and to line 41 at 42, and 3 sets of fluid andtubing detectors (not shown but attached to lines 61, 66 and 41). Duringthe Isolex® 300i procedure the bags 44, 53, 48 and 79 are hung on weightscales 1, 2, 3 and 4, respectively. Bags 74 and 5 are hung together onweight scale 5. Buffer bag 7 is hung on weight scale 5. Buffer bag 7 ishung on weight scale 6. Bags 36, 39 and 30 are not hung on a scale.Weight scale 5 is used to determine the cell product volume in the washcircuit by substracting out the reference weight when the Isoflow™ bagis empty. The weight scales are in the tower of the Isolex® 300iinstrument.

[0051] The stand-alone cell washing system will also run automaticallyusing microprocessors. These microprocessors in turn control 1 bank of 4clamps each, 1 bank of 4 pumps and 1 spinner motor drive. The systemwill require feedback from 4 weight scales, 2 pressure transducers, and3 sets of fluid and tubing detectors.

[0052] The size of the cell mass is minimized by increasing theconcentration ratio (CR) as far as possible. CR is the ratio of the rateof unwashed undiluted cell volume coming into the spinning membranefilter to the rate of washed cell volume exiting the spinning membranefilter. In the wash circuit, there are four variables to control CR, therecirculation rate (r), the buffer solution rate (b), the cell sourcerate (c), and the filtrate rate (f). The relationship is c+b=r+f, and CR=c/r=1+(f−b)/r.

[0053] For both the Isolex® 300i and the Stand-alone system, the cellsare concentrated and washed automatically. We have found that byconcentrating, diluting, and concentrating again multiple times, thevolume can be more consistently controlled. Thus, between every othercell product cycle through the spinner (i.e., spinning membrane filter)the cell volume is diluted and reconcentrated. If the number of cyclesleft is predicted to be less than 2.5 cycles, the dilutions stop. Duringdilutions, the filtrate pump P4 is stopped, the buffer pump P2 runs at afixed rate and the recirculation pump P3 runs at about 110% of thebuffer rate. This allows the membrane to be rinsed and dilutes the cellconcentrate through the port with the more concentrated cells.

[0054] The transmembrane pressure is regulated by controlling theconcentration ratio CR using the recirculation pump P3. Theconcentration ratio CR is contolled to a target pressure by a PID(Proportional/Integrative/Derivative) control through the pressuremeasurements. The pressure measurements are taken from the pressuretransducer connected to the filtrate line and are adjusted for thecentrifugal effects on the fluid to yield a trans-membrane pressure. Ifthe bag volume drops below the target volume, CR is no longer thecontrolling parameter. Instead, the scale weight is controlled by thebuffer pump P2 and CR is calculated as: CR=c/r. Given CR, therecirculation rate is calculated as r=70/CR−1 where CR is limited to>=2.

[0055] Filtrate rate (f) is set to its maximum in order to minimize thetime to process the cells. Filtration pressure is an indicator of theconcentration of blood cells along the membrane of the spinning membranefilter. However, if either the spinner 6, buffer pump P2 orrecirculation pump P3 are not up to speed, the filtrate rate is reduced.The ratio of the measured spinner 6, buffer pump, or recirculation pumprate to the respective commanded rate is calculated. The filtrate rateis then calculated as f₁=¾*MRR*TFR+¼*TFR, where f₁ is the minimum ratioadjusted rate to be commanded in ml/min, MRR is the minimum rate ratiosdescribed above, TFR is the target filtrate rate (70 ml/min). Thefiltrate rate is further reduced when the pressure error (E_(p))described above is less than −5 mmHg. When this condition is true thefiltrate rate is set to f₂=f₁+E_(p)+5, where f₂ is the final commandfiltrate rate and f₁ is the minimum ratio adjusted filtrate ratedescribed above. During dilutions, the filtrate rate is set to 0.

[0056] Recirculation rate (r) is the primary regulating variable. Thebuffer solution rate (b) is used to regulate the concentration ratio CRbetween values of 1 and 2. The buffer pump P2 provides the primaryregulation to the scale weight management control. When the Isoflow™ bag5 fluid volume weight drops below the target (20-35 ml), the buffer iscommanded to about 78 ml/min. This is approximately 8 ml/min faster thanthe filtrate pump P4. This causes the bag weight to rise. Once theweight rises about 5 ml, the buffer once again becomes secondary to theconcentration ratio control, the buffer pump P2 is regulated accordingto the equation b=(70+f)/2−r*(CR−1).

[0057] Because the blood cells can be damaged by stress, the contollerautomatically adjusts the rotor spin rate of the spinning membranefilter. As the recirculation rate (r) is decreased the exposure time ofthe cells in the spinning membrane filter increases as follows:t=v/(r+f), where t and v are time and volume, respectively, in thespinning membrane. When r slows, stress on the cells increases. Thecontroller counteracts this by decreasing the spin rate linearly when ris reduced.

[0058] The amount of washing is based on an estimate of “residual”. Theresidual represents the target component for reduction (e.g., platelets,antibody). This estimate is made possible by the mixing properties ofthe IsoFlow™ bag. The estimate is calculated similar to how serialdilutions would calculate the residual. However, it is recalculatedseveral times a second. The equation is

FSR _(i) =FSR _(i−1)−(F _(i)/(B _(i) +C _(i))×(C _(i) /V _(i))×FSR_(i−1) ×TA

[0059] where i=the discrete time interval

[0060] FSR_(i)=Fraction of Starting Residual at time t_(i)

[0061] FSR₁=Fraction of Starting Residual at time t_(i−1)

[0062] F_(i)=Filtrate volume moved at rate f measured at time intervali−1 to i in units of ml

[0063] B_(i)=Buffer volume moved at rate b measured at time interval i−1to i in units of ml

[0064] C_(i)=Cell source moved at rate c measured at time interval i inunits of ml, including the rate from the IsoFlow™ bag 5, as well as therate of addition of unwashed cells, if any, in same units

[0065] V_(i)=cell product volume at time interval i in ml

[0066] TA=Target Admittance

[0067] The Target Admittance is the unitless constant that representsthe ease with which a given substance passes through the membrane (theinverse of membrane impedance). For platelet wash the Target Admittancehas been found to be between 0.5 and 1.0 with a preferred setting of0.7. For antibody and release agent wash the Target Admittance has beenfound to be between 0.7 and 1.2 with a preferred setting at 1. Theoptimal level for the antibody used for CD34⁺ selection on the Isolex®300i has been found to be in the range of 50-150 micrograms.

[0068] An estimate of the average number of times a cell has beenthrough the spinning membrane acts as a backup for determining when toend a wash. Cell cycles are estimated based on the following equation:

Cell cycles _(i)=∫(R_(j) +F _(j) −B _(j))/V _(j) =∫C _(j) /V _(j)

[0069] where

[0070] R_(j)=Recirculation volume moved at rate r measured at timeinterval j in units of ml, and Cell cycles_(i)=Number of cycles throughthe spinning membrane device that the cell product has experienced attime interval i.

1. A method of recirculation washing of blood cells which utilizes a flexible plastic recirculation wash bag or reservoir having a top port and a bottom port in conjunction with a spinning membrane filter having an inlet port for a diluted suspension of blood cells in buffer solution, a first outlet port for filtrate and a second outlet port for a concentrated suspension of blood cells in buffer solution, which comprises withdrawing a suspension of blood cells in buffer solution from the recirculation wash bag through the top port, mixing the suspension with additional buffer solution to form a diluted suspension of blood cells in buffer solution, feeding the diluted suspension into the spinning membrane filter through the inlet port, withdrawing filtrate comprising buffer solution from the spinning membrane filter through the first outlet port, withdrawing a concentrated suspension of blood cells in buffer solution from the spinning membrane filter through the second outlet port, feeding the concentrated suspension into the bag through the bottom port, and continuing the recirculation washing until the desired amount of washing has been achieved.
 2. The method of claim 1 wherein the suspension of blood cells withdrawn through the top port of the recirculation wash bag is mixed with unwashed blood cells as well as buffer solution before feeding the diluted suspension into the spinning membrane filter.
 3. The method of claim 2 wherein the unwashed blood cells include platelets, the filtrate comprises a suspension of platelets in buffer solution, and the recirculation washing is continued until the platelet content of the concentrated suspension of cells has been reduced to the desired level.
 4. The method of claim 1 wherein the recirculation wash bag at the beginning of the recirculation wash procedure contains, in addition to blood cells, an antibody which specifically binds an antigen on certain of the blood cells which, the filtrate comprises a suspension of the antibody in the buffer solution, and the recirculation washing continues until the concentrated suspension of cells contains a desired level of free of excess, unbound antibody.
 5. The method of claim 1 wherein washing is continued until the fraction of starting residual has reached a predetermined value as determined by the equation: FSR _(i) =FSR _(i−l)−(F _(i)/(B _(i) +C _(i))×(C _(i) /V _(i))×FSR _(i−1) ×TA where i=the discrete time interval FSR_(i)=Fraction of Starting Residual at time t_(i) FSR_(i−1)=Fraction of Starting Residual at time t_(i−1) F_(i)=Filtrate volume moved at rate f measured at time interval i−1 to i in units of ml B_(i)=Buffer volume moved at rate b measured at time interval i−1 to i in units of ml C_(i)=Cell source moved at rate c measured at time interval i in units of ml, including the rate from the IsoFlow™ bag 5, as well as the rate of addition of unwashed cells, if any, in same units V_(i)=cell product volume at time interval i in ml TA=Target Admittance, and residual is the component which the cell washing is targeted to reduce.
 6. A flexible plastic bag or reservoir for recirculation washing of blood cells which has a top port and a bottom port and an integral coarse filter comprising a tube of semi-rigid plastic mesh extending from the top port into the bag.
 7. A flexible plastic bag or reservoir for recirculation washing of blood cells which has a top port and a bottom port and a bubble trap at the top which comprises plastic tubing extending into the bag from the top port.
 8. A flexible plastic bag for recirculation washing of blood cells which has a top port and a bottom port, an integral coarse filter comprising a tube of semi-rigid plastic mesh extending from the top port into the bag and having a closed bottom end and a bubble trap at the top which comprises plastic tubing extending from the top port into the bag inside the mesh tube.
 9. Bag of claim 8 wherein the mesh tube is sufficiently rigid that, when vacuum is pulled on the bag, causing it to collapse, the mesh tube holds an open path in the bag, so that blood cells in a buffer solution entering the bottom port can move up to the top port.
 10. A disposable set for recirculation washing of blood cells comprising a recirculation wash bag which has a top port and a bottom port, a spinning membrane filter which has an inlet port for a diluted suspension of blood cells in buffer solution, a first outlet port for filtrate and a second outlet port for a concentrated suspension of blood cells in buffer solution, and a filtrate bag, plus associated tubing, including tubing for a buffer solution bag, wherein plastic tubing connects the top port of the recirculation wash bag to a mixing zone, plastic tubing with a buffer bag spike coupler at one end is connected to the same mixing zone, the mixing zone is connected by plastic tubing to the inlet port of the spinning membrane filter, the first outlet port of the spinning membrane filter is connected by plastic tubing to the inlet port of the filtrate bag, and the second outlet port of the spinning membrane filter is connected by plastic tubing to the bottom port of the recirculation wash bag.
 11. A disposable set of claim 10 wherein the recirculation wash bag has an integral coarse filter comprising a tube of semi-rigid plastic mesh extending from the top port into the bag.
 12. A disposable set of claim 10 wherein the recirculation wash bag has a bubble trap at the top which comprises plastic tubing extending into the bag from the top port.
 13. A disposable set of claim 10 wherein the recirculation wash bag has an integral coarse filter comprising a tube of semi-rigid plastic mesh extending from the top port into the bag and having a closed bottom end and a bubble trap at the top which comprises plastic tubing extending from the top port into the bag inside the mesh tube.
 14. The disposable set of claim 10 which also includes other bags and associated tubing for use in a magnetic cell selection instrument, including a bag for antibody suspension in buffer, a bag for peptide release agent solution in buffer, a bag for a suspension of selected cells in buffer solution, and a bag for a suspension of non-selected cells in buffer solution. 